This invention is directed to a genetic sequence that has been identified as the locus of mutations that cause epilepsy and to methods for the diagnosis of this disease and for the detection of the presence of the mutated gene as an indication of potential for genetic transmission of a disease.
Cystatin B is a small protein that is a member of a superfamily of cysteine protease inhibitors, which are generally grouped into three families based on structural relationshipsxe2x80x94stefins (of which cystatin B is a member), cystatins and kininogens (Jarvinen, M. et al., Biochim. Biophys. Acta 708:210-217 (1982); Turk, V. et al., FEBS Lett 285:213-219 (1991)). It is a tightly-binding reversible inhibitor of cathepsins L, H and B, is found in all tissues, and is thought to inactivate proteases that leak out of the lysosome (Jarvinen, M. et al., (1982); Turk, V. et al., (1991); Ritonja, A. et al., Biochem. Biophys. Res. Commun. 131:1187-1192 (1985); Jerala, R. et al., FEBS Lett 239:41-44 (1988)). Its amino acid sequence in humans is known (Jarvinen, M. et al., (1982); Turk, V. et al., (1991)). Another member of this family of protease inhibitors, cystatin C, has been shown to be responsible for hereditary cerebral amyloid angiopathy (Abrahamson, M. et al., Hum. Genet. 82:223-226 (1989); Ghiso, J. et al., Proc. Natl. Acad. Sci. U.S.A. 83:2974-2978 (1986)). This dominantly-inherited disease is characterized by the deposition of cystatin C-rich amyloid fibrils in affected brain arteries.
Progressive myoclonus epilepsy refers to a heterogeneous group of severe inherited epilepsies that are characterized by myoclonic seizures, generalized epilepsy and progressive neurological deterioration, including dementia and ataxia (Berkovic, S. F. et al., N. Enql. J. Med. 315:296-305 (1986)). One of the five recognized members of this class is progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1; H. Unverricht, Die Myoclonie (Franz Deuticke, Vienna), pp. 1-128 (1891); H. Unverricht, Disch, Z. Nervenheilk 7:32-67 (1895); H. Lundborg, Die Proqressive Mvoclonus-Epilepsie (Unverrichts Myoklonie) Almquist and Wiksell, Uppsala, pp. 1-207 (1903). This form of epilepsy is inherited as an autosomal recessive disease in which patients have severe stimulus-sensitive myoclonus and tonic-clonic seizures beginning between ages 6 and 15, and have a variable rate of progression between and within families (Koskiniemi, M., Epilepsia 15:537-545 (1974); Koskiniemi, M. et al., Acta Neurol. Scandinav. 50:333-359 (1974); Koskiniemi, M. et al., Acta Neurol. Scandinav. 5:307-332 (1974); Norio, R. et al., Clin Genet. 15:382-384 (1979)). Seizures tend to diminish at 25-30 years of age, although mild dementia generally develops late in the course of the disease. Unlike the other progressive myoclonus epilepsies, inclusion bodies or storage material are not observed in EPM1, and diagnosis of the disease is usually based on clinical history, typical electroencephalographic abnormalities, and the exclusion of the other four subtypes (Lafora""s disease, MERRF syndrome, neuronal ceroid lipofuscinosis and sialidosis); EPM1 and other forms of epilepsy affect about 3% of the world""s population (Scheuer, M. L. et al., N. Enql. J. Med. 323:1468-1474 (1990)).
The genes responsible for Lafora""s disease and juvenile myoclonus epilepsy, which have symptoms similar to EPM1, have been localized to specific chromosomal regions by meiotic linkage analysis (Serratosa, J. M. et al., Hum. Mol. Genet. 4:1657-1663 (1995); Greenberg, D. A. et al., Am. J. Med. Genet. 31:185-192 (1988); Liu, A. W. et al., Am. J. Hum. Genet. 57:68-381 (1995)). Linkage analysis initially localized the gene responsible for EPM1 to a 2 million base pair (Mb) region on the long arm of human chromosome 21 between the DNA markers CBS and CD18 (Lehesjoki, A. E. et al., Proc. Natl. Acad. Sci. U.S.A. 883696-3699 (1991); Lehesjoki, A. E. et al. Neurolocv 42:1545-1550 (1992); Lehesjoki, A. E. et al., Hum. Molec. Genet. 2:1229-1234 (1993)). However, prior to the current elucidation of a molecular defect for cystatin B, no specific defects have been found to be associated with EPM1.
One aspect of the invention is an isolated nucleic acid molecule, wherein said molecule comprises:
(1) a first sequence consisting of human cystatin B genomic DNA as set forth in FIG. 3 (SEQ ID NO:1);
(2) a second sequence, wherein said second sequence is a subsequence of said first sequence, is at least 10 nucleotides in length, and is not present in human cystatin B cDNA;
(3) a third sequence in which at least one nucleotide of said first or second sequences is replaced by a different nucleotide; or
(4) a fourth sequence complementary to any of said first, second or third sequences;
with the proviso that (I) if said molecule is an RNA molecule, U replaces T in said sequence of said molecule, and (ii) said third sequence is at least 95% identical to said first or second sequence.
Another aspect of the invention is an isolated nucleic acid molecule, wherein said molecule comprises a first sequence in which at least one nucleotide of cystatin B cDNA is replaced by a different nucleotide or a second sequence complementary to said first sequence, with the proviso that (I) if said molecule is an RNA molecule, U replaces T in said sequence of said molecule and (ii) said first sequence is at least 95% identical to cystatin B cDNA.
Another aspect of the invention is a method of detecting the presence of a genetic defect that causes epilepsy in a human or that can transmit epilepsy to an offspring of said human which comprises:
identifying a mutation of a cystatin B gene of said human, wherein said mutation provides a cystatin B gene sequence different from human cystatin B genomic DNA sequence as set forth in FIG. 3 (SEQ ID NO:1).
Another aspect of the invention is a method for treating an individual with progressive myoclonus epilepsy, comprising supplying to said individual an effective amount of a gene product of a cystatin B gene.